Process for production of diketopiperazine dihydroxamates and intermediates therefor

ABSTRACT

A PROCESS FOR THE PRODUCTION OF DIKETOPIPERAZINE DIHYDROXAMATES WHICH ARE USEFUL IN THE BIOLOGICAL FIELD AND INTERMEDIATES FOR THE SYNTHESIS OF SAID DIKETOPIPERAZINE DIHYDROXAMATES IS DISCLOSED.

United States Patent US. Cl. 260112.5 1 Claim ABSTRACT OF THE DISCLOSUREA process for the production of diketopiperazine dihydroxamates whichare useful in the biological field and intermediates for the synthesisof said diketopiperazine dihydroxamates is disclosed.

This invention relates to a process for the production of piperazinederivatives. More particularly, it relates to a process for theproduction of cyclo-di-N -acetyl-N -hydroxy-L-ornithyl, i.e-.,diketopiperazine dihydroxamates, and the novel intermediates for thesynthesis of diketopiperazine dihydroxamates and a process for theproduction of such intermediates.

The diketopiperazine dihydroxamates prepared in accordance with theprocess of this invention are represented by the following formula T/COR wherein R represents a lower alkyl group having 1 to 4 carbonatoms, inclusive. The synthesis of compounds of this type has not beenreported in literature, and it was confirmed that this type of compoundexists only in the natural field. For example, rhodotorulic acid whichis included in the product of this invention (R=methyl) was reported byC. L. Atkin et a1., Biochemistry, 7, No. 10, 3734 (1968) in whichrhodotorulic acid was isolated from the fermentation broth of a redyeast identified as Rhodotorula pilimanae and the structural formula ofrhodotorulic acid was determined as cyclo-di-N- acetyl-N-hydroxy-L-ornithyl. It was also reported in Biochemistry, 7, No. 10,3734 (1968) that rhodotorulic acid exhibits a biological activitycomparable to that of schizokinen in Lankfords Bacillus system and thatit also exhibits an activity as a growth factor in the assay usingArthrobacter species. Thus, rhodotorulic acid is an extremely usefulcompound in the bilogical field and the success in synthesis ofrhodotorulic acid is of significant importance in industry.

This invention also contemplates certain novel peptides which are usefulnot only as intermediates for the synthesis of cyclo-di-N"-acetyl-N-hydroxy-ornithyl, but also are biologically active. Peptides isolatedfrom the metabolites of microorganisms frequently contain, as theircomponents, w-hydroxyamino-ix-aminoacids, and therefore the novel aminoacid derivatives represented by the Formula III below are expected to beuseful compounds for various purposes such as the synthesis of a widevariety of biologically active compounds.

The object of this invention is therefore to provide a process for theproduction of diketopiperazine hydroxamate derivatives.

Another object of this invention is to provide novel intermediates forthe synthesis of diketopiperazine hydroxamate derivatives and a processfor the production of such intermediates.

The process according to the present invention can be illustrated by thefollowing reaction scheme:

CH 0 SO --0 3 Q I RN- 11-0 0 OH @cm-o ya 0 4311: (CH2): HiN-H-c 00B.(II) (o t) (CH2): RHN-CH 00 NH o11oo0R (III) @4310 -N-cH,-cm ornQ-s 0r-N-O Gin-Q (6H2): I Da HgN- H 00 NE CH-COOR (W H H N GHr'O N H COR (VI)N 0 OCHT- N COR (VII) N I 0 /COR 110 N OH H vnr wherein R represents aprotecting group as defined here- 35 vent may preferably be acetic acidper se or an acetate inafter, and R is a lower alkyl group having 1 to 4carbon atoms, inclusive.

The starting materials of the process of this invention,N-tosyl-N-benzyloxy-L-ornithine having the Formula I and N-tosyl-N-benzyloxy-L-ornithine alkyl ester having the Formula II, can bederived from w-(N-tosyl-N-benzyloxy)-ornithine which is, in turn,obtainable by hydrolysis of thea-acylamino-w-(O,N-disubstituted-hydroxy)-alkylmalonic acid esterdisclosed in Japanese patent publication No. 24,763/ 1970 proposed bythe present inventors. More specifically, the starting material havingthe Formula I can be produced by first subjecting the above N -tosyl-N-benzyloxy-DL-ornithine to optical resolution to obtain thecorresponding L-amino acid and then protecting the tZ-amiHO group of theL-amino acid with a protecting group. The term protecting group usedherein means any one of the conventional protecting groups for the aminogroup commonly employed in the art of peptide synthesis and includes,for example, a carbobenzoxy group, a t-butoxycarbonyl group, apmethoxybenzyloxycarbonyl group and the like. Another starting materialhaving the Formula II (a lower alkyl ester) can easily be produced fromthe above L-amino acid by the conventional ester formation technique,for example, by suspending the L-amino acid in an appropriate alcoholand passing hydrogen chloride gas into the resulting alcoholicsuspension.

The reaction between the starting materials (I and II) can beaccomplished by coupling the materials in an approximately molarequivalent in accordance with the acid anhydride method or theisoxazoline method in a solvent such as tetrahydrofuran, nitromethane orthe like, at a temperature in the range of from C. to 10 C. therebyyielding an L-hydroxyornithine dipeptide derivative represented by theFormula III. The subsequent removal of the protecting group from theresulting compound (III) can be carried out by any one of the well-knownprocedures, such as by treating the compound (III) while stirring in anacetic acid solvent in the presence of a hydrogen halide. The aceticacid solester. The removal of the protecting group can be conducted atroom temperature, i.e., a temperature between about C. and C. Under suchconditions, the desired dipeptide ester having the Formula IV can beobtained in the form of its hydrohalide salt in high purity and highyield. It is to be understood that the above coupling reaction and theremoval of protecting group may also be adapted to the correspondingD-forrn thereby resulting in the production of the desired compound (IV)in the D-form.

The cyclization of the thus obtained dipeptide ester of the Formula IVcan then be conducted in an appropriate alcohol using ammonia gas toproduce cyclodi-N -tosyl-N -benzyloxy-ornithyl of the Formula V. Forexample, the cyclication may be carried out by adding the dipeptideester in the form of its hydrohalide salt to methanol which haspreviously been saturated with anhydrous ammonia and allowing themixture to stand for several days at a temperature of from about 0 C. toroom temperature.

The resulting compound (V) is then subjected to detosylation procedureto give a novel compound having the Formula VI,cyclo-di-N"-benzyloxy-ornithyl. The detosylation can be eflected bytreatment of the compound of the Formula V in a specific solvent system.The solvent system used in the detosylation step comprises hydrogenbromide, acetic acid and phenol in such a proportion that hydrogenbromide in acetic acid is a solution containing hydrogen bromide in anamount of to by weight, preferably about 36% by weight and that phenolis used in an amount of from 0.1 to 0.3 parts by weight per 1 part byweight of acetic acid. The detosylation can be accomplished by addingthe compound (V) to the above hydrogen bromide-acetic acid-phenolsolvent system and stirring the mixture at approximately roomtemperature, generally in the range of from 18 C. to 25 C., for at least30 hours, generally from 30 to hours. At lower temperatures the reactiondoes not proceed smoothly, while at higher temperatures the desireddetosylated compound (VI) tends to decompose. Unduly prolonged stirringalso results in the decomposition of the desired compound (VI). Aftercompletion of the reaction, the compound (VI) can be isolated ascrystals from the reaction mixture by any one of the well-knowntechniques. It is also to be understood that the detosylation proceduremay be adapted to the compound (V) in either D-, L- or DL-form therebyyielding the corresponding optical isomer ofcyclo-di-N-benzyloxyornithyl having the Formula VI.

The thus obtained compound (VI) can then be acylated with an acidanhydride of lower aliphatic acids in a basic solvent to give cyclo-di-"-acetyl- -benzyloxyornithyl having the Formula VII. The acetylation caneasily be conducted by any one of the well-known acylation procedures,and the solvent of choice for the acylation includes quinoline andpyridine. Again, the acylation may be employed for the compound (VI) ineither D, L- or DL-form thereby yielding the corresponding opticalisomer of the acylate (VII).

The final step of the process of this invention is debenzylation of theabove acylate (VII). This debenzylation can be carried out by catalyticreduction in the presence of a catalyst, for example,palladium-on-carbon, and the like, at normal temperature under normalpressure. The amount of the catalyst is not critical and may be aso-called catalytic amount. The debenzylation is usually completedwithin about 10 to 20 hours at normal temperature. The desired finalproduct of the Formula VIII, cyclo-di-N-acetyl-N -hydroxy-ornithyl, canbe isolated from the reaction mixture in a usual manner and purified byrecrystallization, etc. The debenzylation is also employed for thecompound (VII) in either D-, L- or DL-form thereby yielding thecorresponding optical isomer of the product having the Formula VIII.

The present invention is further illustrated by the following examples,but they are not to be construed as limiting the scope of the invention.

EXAMPLE 1 (A) N -benzyloxycarbonyl N tosyl N-benzyloxy-L- ornithyl--tosyl-N benzyloxy L ornithine methyl ester 1.26 ml. of isobutylchloroformate was added to a cooled solution of 1.31 ml. oftriethylamine and 4.90 g.

of N benzyloxycarbonyl-N -tosyl-N -benzyloxy-L-ornithine in 45 ml. oftetrahyorofuran at a temperature of from-10 to C. After minutes, to theresulting solution was added a solution of 4.24 g. of N-tosyl-N"-benzyloxy-L-ornithine methyl ester hydrochloride and 1.31 ml. oftriethylamine. The mixture was then stirred overnight at roomtemperature, and evaporated in vacuo. The residue was combined withwater, and the mixture was filtered to collect the product. The thusobtained product was dissolved in ml. of ethyl acetate, and the solutionwas washed successively with 1 N hydrochloric acid, water, 0.5 M aqueoussodium bicarbonate and water and dried over anhydrous sodium sulfate.The solution was then filtered and evaporated in vacuo to dryness togive a product identified as N"-benzyloxycarbonyl-N- tosyl-N-benzyloxy-L-ornithyl N" tosyl-N -benzyloxy- L-ornithine methyl ester byits N.M.R. and LR. spectra. Yield: 8.4 g. (98%).

Analysis.Calcd. for C H .,N O S (percent): C, 61.70; H, 5.90; N, 6.13.Found (percent): C, 61.59; H, 6.12; N, 6.25.

(B) N -tosyl-N -benzyloxy-L-ornithyl-N"-benzyloxy- L-ornithine methylester hydrobromide 8.2 ml. of 36% hydrogen bromide-acetic acid was addedto a solution of 8.3 g. of N-benzyloxycarbonyl-"-tosyl-N-benzyloxy-L-ornithyl N tosyl -benzyloxy-L-ornithine methylester obtained in Example 1(A) in 6.5 ml. of acetic acid. After allowingto stand for 90 minutes, the resulting solution was evaporated in vacuo,

and the residual oily substance was triturated with ether and repeatedlywashed with ether by decantation. The resulting oily substance wasidentified as N"-tosyl-N-benzyloxy-L-ornithyl-N tosyl Nbenzyloxy-L-ornithine methyl ester hydrobromide by its thin layerchromatography. Yield: 6.3 g. (79%).

EXAMPLE 2 (A) N -t-butyloxycarbonyl N -tosyl N benzyloxy-L- ornithyl-Ntosyl N benzyloxy-L-ornithine methyl ester 3.82 ml. of triethylamine wasadded to a suspension of 12.26 g. of N-t-butyloxycarbonyl- -tosyl-N'-benzyloxy- L-ornithine and 6.31 g. of N-ethyl-5-phenylisoxazolium3'-sulfonate in 80 ml. of nitromethane charged into a sealed glassvessel at a temperature of 0 C. The mixture was stirred in an ice bathuntil a clear solution was obtained, i.e., for two hours. To theresulting solution was then added 11.30 g. ofN-tosy1-N-benzyloxy-L-ornithine methyl ester hydrochloride and 3.82 ml.of triethylamine at a temperature of 0 C. The reaction mixture wasstirred for two hours at 0 C. and then overnight at room temperature,and evaporated in vacuo to give an oily substance. The thus obtainedresidue was extracted with ethyl acetate, and the ethyl acetate extractwas washed with water, an aqueous sodium carbonate (three times) andfinally water followed by drying over magnesium sulfate. Afterfiltration, the filtrate was evaporated in vacuo, and the oily residuewas triturated with petroleum ether. The thus obtained product waspurified by dissolving in ethyl acetate and adding petroleum ether togive a purified product identified as N-t-butyloxycarbonyl-N -tosyl-N-benzyloxy-L-ornithyl-N"-tosyl-N benzyloxy-L-ornithine methyl ester byits N.M.R. and LR. spectra. Melting point, 63-69 C.; [a] +1.1 (c., 1 inethyl acetate).

Analysis.-Calcd. for C H N O S (percent): C, 59.98; H, 6.41; N, 6.37.Found (percent): C, 60.09; H, 6.65; N, 6.10.

(B) N tosyl N benzyloxy L ornithyl N tosyl- N"-benzyloxy-L-ornithinemethyl ester hydrochloride 80 ml. of 3 N hydrochloric acid in ethylacetate was added to a solution of 18.76 g. of N-t-butyloxycarbonyl- N-tosyl-N -benzyloxy L ornithyl-N -tosyl-N-benzyloxy-L-ornithine methylester obtained in Example 2(A). The resulting solution was allowed tostand at room temperature for two hours and evaporated in vacuo to givean oily substance which was then triturated with ether to crystallize.The thus obtained crystalline product was identified as N -tosyl-N-benzyloxy-L-ornithyl-N- tosyl-N -benzyloxy-L-ornithine methyl esterhydrochloride by its N.M.R. and LR. spectra. Yield: 16.10 g. (92.5%).Melting point, IOU- C., [u] +l2.10 (c, 1 in methanol).

Analysis.-Calcd. for C H N O S Cl (percent): C, 57.30; H, 6.04; N, 6.85.Found (percent): C, 56.98; H, 6.24; N, 6.58.

EXAMPLE 3 (A) N,-t-butyloxycarbonyl-N -tosyl-N -benzyloxy-D-ornithine-N-tosyl-N -benzyloxy D-ornithine methyl ester The reaction described inExample 2(A) was carried out under the same conditions but usingN-t-butyloxycarbonyl-N -tosyl N benzyloxy D ornithine and N- tosyl N"benzyloxy-D-ornithine methly ester hydrochloride in place of N-t-butyloxycarbonyl- "-tosyl-N"- benzyloxy-L-ornithine andN"-tosyl-N-benzyloxy-I..-ornithine methyl ester hydrochloride,respectively, to obtain N"-t-butyloxy-carbonyl-N -tosyl-N-benzyloXy-D-ornithyl- N"-tosyl-N-benzyloxy-D-ornithine methyl esterhaving a melting point of 63-68" C. Yield: 90.3%, [a] -l.4 (c., 1 inethyl acetate).

Analysis.-Calcd. for C H N O S (percent): C, 59.98; H, 6.41; N, 6.37.Found (percent): C, 60.01; H, 6.69; N, 6.03.

7 (B) N-tosyl-N"-benzyloxy D ornithyl N tosyl-N- benzyloxy-D-ornithinemethyl ester hydrochloride The reaction described in Example 2(B) wascarried out under the same conditions but using N-t-butyloxycarbonyl-N-tosyl-N -benzyloxy D ornithyl-N-tosyl-N- benzyloxy-D-ornithine methylester to obtain N"-tosyl-N- benzyloxy D ornithyl N" tosyl N" benzyloxy-D-ornithine methyl ester hydrochloride having a melting point of 102l07C. Yield: 82.8%, [a] -11.4 (c., l in methanol).

Analysis.Calcd. for C H N O S (percent): C, 57.30; H, 6.04; N, 6.85.Found (percent): C, 57.33; H, 6.08; N, 6.61.

EXAMPLE 4 (A Cyclo-di-N"-tosyl-N-benzyloxy-L-ornithyl 3.95 g. ofN"-tosyl-N-benzyloxy-L-ornithyl-N"-tosyl- N-benzyloxy-L-ornithine methylester hydrochloride prepared as in Example 1(B) was added to 50 ml. ofmethanol previously saturated with anhydrous ammonia at a temperature ofC., and the mixture was allowed to stand in a glass vessel for two daysat room temperature. The crystals (1.1 g.) formed during the standingwere filtered, and the filtrate was concentrated to dryness. Theresulting crystalline residue was combined with water followed byfiltration, and recrystallized from methanol to give cyclo di N tosyl-N-benzyloxy-L- ornithyl. Yield: 54%. Melting point, 176-177 C.,

[u] 3l.5 (in dimethylformamide) Analysis.Calcd. for C H N O S (percent):C, 60.94; H, 5.92; N, 7.48; S, 8.56. Found (percent): C, 61.15; H, 5.85;N, 7.48; S, 8.59.

(B) Cyclodi-N benzyloxy-Dornithyl dihydrobromide 1.88 g. ofcyclo-di-N"-tosyl-N-benzyloxy L- ornithyl prepared as in Example 4(A)was added to a solution of 5 g. of phenol in 20 ml. of 36% hydrogenbromide-acetic acid at room temperature. After stirring for 50 hours atroom temperature, the mixture was evaporated in vacuo 8 EXAMPLE 6 (A)Cyclo-di-N-acetyl-N-benzyloxy-L-ornithyl 0.78 g. ofcyclo-di-N"-benzyloxy-L-ornithyl prepared as in Example 4(B) was mixedwith 6.5 ml. of pyridine and 6.5 ml. of acetic anhydride. The resultingmixture was stirred for 20 hours at room temperature and then evaporatedin vacuo. The thus obtained residue was combined with water and themixture was evaporated (this treatment being repeated twice) todecompose any remaining acetic anhydride present in the mixture in asmall amount. The resulting residue was taken into 30 ml. of ethylacetate, and the solution was washed repeatedly with water. The organiclayer was dried over sodium sulfate and evaporated in vacuo to dryness.Recrystallization of the thus obtained residue from ethanol-etheryielded cyclo-di-N -acetyl-N -benzyloxy-L-ornithyl having a meltingpoint of 9799 C. Yield: 68%. [06 16.4 (c., 1 in ethanol).

Analysis.-Calcd. for C H N O (percent): C, 64.10; H, 6.92; N, 10.68.Found (percent): C, 63.97; H, 6.86; N, 10.62.

(B) Cyclo-di-N"-acetyl-N -hydroxyl-ornithyl 0.46 g. ofcyc1o-di-N-acetyl-N-benzyloxy-L-ornithyl in 40 ml. of ethanol washydrogenated in the presence of 0.75 g. of 5% palladium-on-carbon atroom temperature. After 15 minutes, the reaction mixture was filtered toremove the catalyst, and the filtrate was evaporated in vacuo at roomtemperature. Recrystallization of the residual crystals from boilingwater yielded 0.2 g. (66% yield) of the product having a melting pointof 217-2l8.5 C. (with decomposition). [M 28.5 (c., 1 in water). The thusobtained product gave a positive ferric chloride reaction (red-purplecolor) and was identified as cyclo-di-N -acetyl- N-hydroxy-L-ornithyl(rhodotorulic acid) by its N.M.R. and LR. spectra shown in Table 1below.

Analysis.Calcd. for C H N O (percent): C, 48.83; H, 7.03; N, 16.27.Found (percent): C, 48.81; H, 7.07; N, 16.18.

TABLE 1.N.M.R. AND LR. SPECTRA OF PRODUCT at a temperature of 30 C., andthe residual oil substance N.M.R I was crystallized by adding ether. Theresulting crystals I were filtered and washed successively with ethanoland (Flccoomm 2. 2 fi j 'figfi g g ether to give the desired product. Aportion of the product g? 51echainnethy1ene adjacent to oge wasrecrystallized from ethanol for analysis to give cyclo- 2 :25 333di-N-benzyloxy-L-ornithyl hydrobromide having a melting point of 183-1860. LR

Analysis.--Calcd. for C H N O -2HBr (percent): C, (Cmr 3,180 1, 40048.01; H, 5.37; N, 9.33. Found (percent): 0, 47.92; H, 3 233 Hgg 5.81;N,9.10. 11090 (Amide I) '060 The product was identified, by its N.M.R. andLR. i228 (Hydmxamm am carbwyl) spectra, as a compound having thefollowing structural 1,222 769 formula:

M o Q o N H EXAMPLE7 EXAMPLE 5 cyclo-di-N -benzyloxy-D-ornithyldihydrobromide Cyclo-di-N -acetyl-N"-hydroxy-D-ornithyl Following theprocedure described in Example 6(B) under the same conditions, but usingcyclo-di-N -acetyl- N"-benzyloxy-D-ornithyl in place of cyclo-di-N-acetyl- N benzyloxy-L-ornithyl, there was obtained cyclo-di-N- acetyl-N-hydroxy-D-ornithyl having a melting point of 216-2185. Yield: 38.8%.[a] +28.0 (c., 0.6 in water).

Analysis.Calcd. for C H N O (percent): C, 48.83; H, 7.03; N, 16.27.Found (percent): C, 48.72; H, 6.88; N, 16.05.

What We claim is: wherein R represents a protecting group and Rrepresents 1. L-hydroxyornithine dipeptide compounds of the fora loweralkyl group having 1 to 4 carbon atoms, inclusive. mula:

No references cited.

ELBERT L. ROBERTS, Primary Examiner US. Cl. X.R. 260-268

